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  Detection of post-translationally modified peptides with liquid crystalsUSPTO Application #: 20060003389Title: Detection of post-translationally modified peptides with liquid crystals Abstract: A method for differentiating between a post-translationally modified peptide and a peptide contained in a sample, comprising: (a) contacting the sample with a peptide attachment surface to create a peptidized surface, wherein the sample includes at least one functional group; (b) contacting the peptidized surface with a recognition reagent that selectively binds or forms a complex with the post-translationally modified peptide in the sample to provide an incubated surface; and (c) contacting a liquid crystal with the incubated surface and detecting presence of post-translationally modified peptide in the sample with the liquid crystal. (end of abstract)
Agent: Godfrey & Kahn, S.c. - Milwaukee, WI, US Inventors: Nicholas L. Abbott, Brian H. Clare, Paul J. Bertics USPTO Applicaton #: 20060003389 - Class: 435007900 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Assay In Which An Enzyme Present Is A Label The Patent Description & Claims data below is from USPTO Patent Application 20060003389. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/581,198, filed on Jun. 18, 2004 and U.S. Non-provisional patent application Ser. No. 10/711,517, filed on Sep. 23, 2004, which in turn claims the benefit of U.S. Provisional Application 60/505,114, filed on Sep. 23, 2003, all of which applications are incorporated herein by reference for all purposes. FIELD OF THE INVENTION [0003] The invention relates generally to methods and devices for differentiating between modified peptides and peptides. More particularly, the invention relates to methods and devices for differentiating between phosphorylated peptides and peptides using liquid crystals. BACKGROUND OF THE INVENTION [0004] Methods for detecting phosphorylation of peptides and proteins is an area in the fields of analytical chemistry, medicinal chemistry, and biochemistry where considerable effort has been expended. Considerable efforts have also been made with respect to methods for detecting other post-translational modifications of peptides and proteins such as acylation, glycosylation, alkylation, and adenylation. [0005] Post-translational modification of proteins has been recognized for decades as a significant mode of regulation. In particular, phosphorylation, and the reverse process, dephosphorylation, are key factors in numerous aspects of cell signaling, cell cycle regulation, and response to stress (reviewed in Yan, J. X. et al., Journal of Chromatography A, 808:23-41 (1998)). Phosphorylation of proteins is catalyzed by a class of enzymes called protein kinases, which transfer the terminal phosphate from adenosine triphosphate (ATP) to a given amino acid residue, typically serine, threonine, or tyrosine. In general, phosphorylation is a reversible process. Dephosphorylation is carried out by protein phosphatases. Moreover, kinases and phosphatases may be inhibited by various factors (Hidaka, H. et al., Biochemistry 23:5036-5041 (1984)). Because of their importance in cell signaling and cell cycle regulation, proteins in the phosphorylation cycle, both enzymes and their substrates, have become major targets for the development of pharmaceutical compounds. Protein kinases, in particular, because of their role in cell division and cancer progression, have emerged as the principal targets of drugs aimed at treating cancer, immunosuppression, retinopathy, rheumatoid arthritis, and neurodegeneration (Cohen, P., Nature Reviews Drug Discovery, 1:309-315 (2002)). [0006] A variety of methods exists for monitoring and detecting the phosphorylation state of proteins, which is important, among other purposes, for assessing the efficacy of candidate pharmaceutical agents. Antibody-based detection is among the most widely used of these methods. In addition to monoclonal antibodies specific for individual proteins, more recent endeavors have resulted in the production of phospho-motif antibodies, which recognize a phosphoserine or phosphothreonine residue in a conserved amino acid motif (reviewed in Berwick, D. C. and Tavare, J. M., Trends in Biochemical Science, 29:227-232 (2004)). Generation of such antibodies requires extensive characterization of the substrate specificity of the kinases being examined. Alternative methods for monitoring kinase activity make use of .sup.32P_radiolabeled phosphate groups and mass spectrometry to identify modification in protein composition before and after treatment with a kinase (Yan, J. X. et al., Journal of Chromatography A, 808:23-41 (1998)). [0007] In addition to phosphorylation, other co- and post-translational modifications are known to exert regulatory effects on proteins. Acylation, particularly by either fatty acyl or prenyl residues being covalently linked to an --SH group of a cysteine residue, is one such modification (Kendrew, J. editor, THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY, Blackwell Science, Inc. Cambridge, Mass., 1994, p. 15). Ras proteins undergo several post-translational modifications, including farnesylation. Inhibition of the enzyme that carries out this modification, farnesyl transferase, is a promising approach to controlling this oncogenic protein (Crul, M., et al., Anticancer Drugs. 12(3):163-84 (2001)). Other commonly encountered post-translational modifications, such as glycosylation and proteolytic cleavage, are important in protein secretion and translocation. [0008] Although various methods have been used to detect phosphorylation and other co- and post-translational modifications of peptides and proteins, a need exists for simple devices and methods that may be used to rapidly detect such modifications, particularly the phosphorylation of peptides and proteins without the need for radioactive labeling and other manipulation, such as hybridization and washing, and without the need for complex instrumentation. A need also remains for methods of manufacturing devices for use in differentiating between post-translationally modified peptides and peptides. Also needed are rapid, high throughput methods for directly detecting phosphorylation state irrespective of the identity of the modified amino acid or its location within a particular protein sequence or known kinase motif. SUMMARY OF THE INVENTION [0009] The present invention provides devices and methods for differentiation between post-translationally modified peptides and peptides using liquid crystals. The invention also provides a method for preparing devices and kits for differentiating between post-translationally modified peptides and peptides. [0010] In one aspect, the invention provides a method for differentiating between a post-translationally modified peptide and a peptide contained in a sample. The method generally comprises: (a) contacting the sample with a peptide attachment surface to create a peptidized surface, where the sample includes at least one functional group; (b) contacting the peptidized surface with a recognition reagent that selectively binds or forms a complex with the post-translationally modified peptide in the sample to provide an incubated surface; and (c) contacting a liquid crystal with the incubated surface and detecting presence of post-translationally modified peptide in the sample with the liquid crystal. [0011] In a preferred embodiment, the method includes: (a) contacting the sample containing a post-translationally modified peptide, a peptide, or a mixture thereof with a peptide attachment surface to create a peptidized surface; (b) contacting the peptidized surface with a recognition reagent that selectively binds or forms a complex with the post-translationally modified peptide if present to provide an incubated surface; and (c) contacting a liquid crystal with the incubated surface. The post-translationally modified peptide, the peptide, or the mixture thereof contained in the sample comprises a functional group selected to react with exposed functional groups on the surface. In preferred embodiments, the peptide attachment surface includes: (i) a support; (ii) a metal deposited on the support providing a metallized surface; and (iii) a functionalized thiol compound bound to the metallized surface, the functionalized thiol compound includes a first thiol compound and a functional group that reacts with the functional group on the post-translationally modified peptide, the peptide, or the mixture thereof when the post-translationally modified peptide, the peptide, or the mixture thereof is contacted with the peptide attachment surface. In preferred embodiments, the functional group of the thiol is a maleimide group and the peptide has a terminal cysyeine residue. As before, the orientation of the liquid crystal is different when the liquid crystal is contacted with the incubated surface when the incubated surface includes the post-translationally modified peptide than the orientation of the liquid crystal is when the liquid crystal is contacted with the incubated surface when the incubated surface does not include the post-translationally modified peptide. In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the peptide attachment surface further includes a second thiol compound that is bound to the metallized surface. [0012] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the method includes post-translationally modifying the peptide after the peptide has been contacted with the peptide attachment surface. [0013] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the functional group on the functionalized thiol compound is a maleimide group. [0014] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the method includes reacting the first thiol compound with a heterobifunctional linker to provide the functionalized thiol compound. In some such embodiments, the first thiol compound has an amine or ammonium group and the heterobifunctional linker is sulfosuccinimidyl-4-(N-maleimidomethyl)cyclo- hexane-1-carboxylate, and the amine or ammonium group of the first thiol compound reacts with the heterobifunctional linker to provide the functionalized thiol. [0015] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the first thiol compound is a compound of formula HS--(CH.sub.2).sub.a--(OCH.sub.2CH.sub.- 2).sub.b--NH.sub.2 or an ammonium salt thereof, wherein a is an integer ranging from 1 to 30, or in some embodiments ranging from 4 to 22, and b is an integer ranging from 0 to 10, or in some embodiments ranging from 1 to 5. In some such embodiments, the first thiol compound is HS--(CH.sub.2).sub.1 (OCH.sub.2CH.sub.2).sub.3NH.sub.2 or an ammonium salt thereof. In some embodiments, the compound is the ammonium chloride salt such as a compound of formula HS--(CH.sub.2).sub.a--(OCH.sub.2CH.sub- .2).sub.b--NH.sub.3.sup.+Cl.sup.-. [0016] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the second thiol compound is a compound of formula HS--(CH.sub.2).sub.c--(OCH.sub.2CH.sub.- 2).sub.d--X, wherein c is an integer ranging from 1 to 30, or in some embodiments ranging from 4 to 22, and d is an integer ranging from 0 to 10, or in some embodiments ranging from 1 to 5, and X is selected from an --OH, an alkoxy group, a CH.sub.3, a sugar, a zwitterionic group, or a polar non-ionic group. In some such embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the second thiol compound is a compound of formula HS--(CH.sub.2).sub.c--(OCH.sub.2CH.sub.2).sub.d--OH, wherein c is an integer ranging from 1 to 30, or in some embodiments ranging from 4 to 22, and d is an integer ranging from 0 to 10, or in some embodiments ranging from 1 to 5. In some such embodiments, the second thiol compound is HS--(CH.sub.2).sub.11(OCH.sub.2CH.sub.2).sub.3OH. [0017] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the molar ratio of the first thiol compound to the second thiol compound on the metallized surfaces of the peptide attachment surface ranges from 0.1:99.9 to 100%. In other embodiments, the molar ratio of the first thiol compound to the second thiol compound on the metallized surfaces of the peptide attachment surface ranges from 0.2:99.8 to 20:80. In still other embodiments, the molar ratio of the first thiol compound to the second thiol compound on the metallized surfaces of the peptide attachment surface ranges from 0.2:99.8 to 5:95, from 0.2:99.8 to 10:90, from 0.5:99.5 to 10:90, or from 0.5:99.5 to 5:95. [0018] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the liquid crystal is a nematic liquid crystal. In some such embodiments, the liquid crystal is 4-pentyl-4'-cynaobiphenyl (5CB). In other embodiments, the liquid crystal is N-(4-methoxybenzylidene)-4-butylaniline (MBBA). In further embodiments, the liquid crystal TL205 (E. Merck, Darmstadt, Germany). [0019] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the metallized surface of the peptide attachment surface has a top layer of gold. In some such embodiments, the top layer of gold has a thickness ranging from 5 nm to 30 nm. In some embodiments, the top layer of gold overlies a layer of a material that promotes adhesion of the gold to the support, which in some embodiments may be titanium. In some such embodiments, the layer of the material that promotes adhesion of the gold is a layer of titanium with a thickness ranging from 0.5 nm to 10 nm. [0020] In some embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the post-translationally modified peptide is a phosphorylated peptide. In some such embodiments, the recognition reagent is an antibody or antibody fragment that selectively binds or forms a complex with the phosphorylated peptide. In other such embodiments, the recognition reagent is a cationic compound. In other embodiments, the recognition reagent is a cationic surfactant, a polyelectrolyte, a cationic iron compound, or a phosphosensor dye that selectively binds or forms a complex with a phosphate group on the phosphorylated peptide. In other embodiments, the phosphorylated peptide has at least one phosphorylated serine and/or phosphorylated threonine residue. In other embodiments, the phosphorylated peptide has at least one phosphorylated tyrosine residue. [0021] In other embodiments of the method for differentiating between a post-translationally modified peptide and a peptide, the post-translationally modified peptide is an acylated, glycosylated, adenylated, farnesylated, or alkylated peptide or is a peptide that has been proteolytically cleaved. Continue reading... Full patent description for Detection of post-translationally modified peptides with liquid crystals Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Detection of post-translationally modified peptides with liquid crystals patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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